Structural determinants of murine leukemia virus reverse transcriptase that are important for template switching, fidelity, and drug-resistance [electronic resource] /

by Svarovskaia, Evguenia S.

Abstract (Summary)

Retroviral reverse transcriptases (RTs) frequently switch templates
within the same RNA (intramolecular) or between copackaged viral RNAs
(intermolecular). Template switching by RT is necessary for completion of
reverse transcription and results in the generation of mutations and
recombination, which increase variation in retroviral populations. The
structural features of retroviral RTs that affect the frequency of template
switching are currently unknown. To identify structural elements of murine
leukemia virus (MLV) RT important for template switching, we developed an in
vivo assay. This assay used a retroviral vector encoding directly repeated
sequences composed of overlapping fragments of the green fluorescent protein
gene (GFP) that was permitted to undergo one round of reverse transcription.
Template switching within the direct repeats functionally reconstituted GFP.
We quantified the effect of mutations in RT on the frequencies of template
switching. Using MLV RTs containing single amino acid substitution mutations
in the Tyr-X-Asp-Asp (YXDD) motif, the dNTP binding site, the thumb domain,
and the RNase H domain were characterized. The results showed that some
mutations in the YXDD motif and dNTP binding site increased the frequency of
template switching, while all mutations in the RNase H domain tested decreased
the same frequency. Hydroxyurea treatment, which depleted nucleotide pools
and reduced the rate of DNA synthesis, increased the frequency of template
switching for the wild-type RT and the YXDD mutant RTs, but not for the
RNase H mutant RTs. Based on these results, we proposed a dynamic copy-
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choice model in which both the rate of DNA polymerization and the rate of
degradation of the RNA template influence the frequency of RT template
switching.